Method and apparatus for controlling a stream of liquid test packages in a capsule chemistry analysis system

ABSTRACT

A method and apparatus for controlling a stream of liquid and air segments wherein the liquid and air segments are selectively aspirated into a first fluid conduit in a plurality of cycles, each cycle beginning with the aspiration of a first air segment and ending with the aspiration of a final air segment. The liquid and air segments are then transferred from the first fluid conduit to a second fluid conduit. The volume of the final air segment of each cycle is then adjusted after the final air segment has moved into the second fluid conduit. Next, the liquid segments and the air segments of each cycle are transferred from the second fluid conduit to a third fluid conduit. The volume of the first air segment of each cycle is then adjusted after the first air segment has moved into the third fluid conduit.

FIELD OF THE INVENTION

This application is a divisional application of U.S. Ser. No.09/111,162, filed Jul. 6, 1998 now U.S. Pat. No. 6,348,354.

The present invention relates to a capsule chemistry sample liquidanalysis system adapted for the automated clinical analysis ofpluralities of human biological sample liquids, in particular to amethod and apparatus for controlling a stream of liquid test packagesflowing through such a system.

BACKGROUND

U.S. Pat. Nos. 5,268,147 (the “'147 patent”) and 5,399,497 (the “'497patent”), owned by the assignee hereof, the disclosures of which areincorporated herein by reference, describe a capsule chemistry sampleliquid analysis system which operates through repeated reversible,bi-directional flow of an appropriately configured stream of liquid testpackages, each test package consisting of alternating segments of aliquid, such as a sample, reagent or buffer, and air, to enablerepeated, precisely timed analysis of the sample of each of the testpackages in the stream by one or more sample liquid analysis means. Asdescribed in the '147 and '497 patents, the system generally comprises asample liquid test package metering and supply means which operates tometer successive test packages for reaction and analysis within thesystem; a reversible direction sample liquid test package displacementmeans which operates to bi-directionally displace the thusly metered andsupplied test packages through the system; a test package transfer meanswhich operates in conjunction with the test package metering and supplymeans and the test package displacement means to provide for thesuccessive test package supply and bi-directional functions of thesystem; test package reaction and analysis means for analysis of thethusly supplied and displaced text packages; and detection meansoperatively associated with the reaction and analysis means to detectand quantify the successive sample liquid test package analysis results.Such a system is particularly adapted to conduct automated clinicalanalysis of pluralities of human biological sample liquids and can beconfigured to perform various specific analyses, including well known socalled chemistry and immunoassay analyses.

An embodiment of the system just described configured for conducting achemistry analysis is shown in FIG. 1A. A second embodiment of thesystem just described configured for conducting an immunoassay analysisis shown in FIG. 1B.

As described in detail in the '147 and '497 patents, the systemgenerally operates by creating a plurality of test packages andsuccessively inserting the test packages into analytical line 30 shownin FIGS. 1A and 1B, which preferably comprises a flexible conduit madeof transparent Teflon or like material. The test packages are thenrepeatedly and bi-directionally flowed back and forth in the analyticalline 30 and ultimately past the appropriate test package reaction,analysis and detection means. Depending on the type of analysis beingdone, e.g., chemistry or immunoassay, particular devices are disposedalong the analytical line 30 so as to comprise the reaction, analysisand detection means. As shown in FIG. 1A, a configuration appropriatefor a chemistry analysis includes first, second and third flow cells 45and vanish zone 50 disposed along the analytical line 30. Thefunctionality of these components is described in greater detail bothbelow and in the '147 and '497 patents. As shown in FIG. 1B, aconfiguration appropriate for an immunoassay analysis includes vanishzone 50, first, second and third bubble detectors 55, first and secondmagnets 60 and luminometer 65. The functionality of these components isdescribed in greater detail both below and in the '147 and '497 patents.

It will be apparent to one of skill in the art that a test packagesuitable for a so-called sandwich type magnetic particle-basedheterogeneous immunoassay is as shown in FIG. 2. In particular, thistest package consists of six liquid segments: a magnetic particlesuspension designated as MP, a mixture of sample and first and secondreagents designated as S/R₁/R₂, a first wash designated as W₁, a secondwash designated as W₂, a combination of a third reagent and a fourthreagent designated as R₃/R₄, and a marker dye designated as D. Asillustrated in FIG. 2, each of these liquid segments is separated by anair segment designated as A₁/A₃, which is a combination of successivelymetered air segments A₁ and A₃, or an air segment designated as A₂. Itwill also be apparent to one of skill in the art that other suitableimmunoassay test packages exist, for example a test package containingeight liquid segments.

It will also be apparent to one of skill in the art that a suitable testpackage for a chemistry analysis is as shown in FIG. 3. In particular,this test package consists of three liquid segments: a second reagentdesignated as R₂, a mixture of sample and a first reagent designated asS/R₁, and a buffer designated as B. As shown in FIG. 3, with theexception of the S/R₁ and R₂ segments, each of these liquid segments isseparated by an air segment designated as A₁, A₂ and A₃. The S/R₁ and R₂segments are separated by a vanish bubble VB comprising air. Asdescribed in detail in the '147 and '497 patents, this vanish bubble VBoperates in connection with vanish zone 50 shown in FIG. 1A to cause amixing of the S/R₁ and R₂ segments at an appropriate time.

Referring again to FIGS. 1A and 1B, the sample liquid test packagemetering and supply means which creates the test packages describedabove includes an aspirating probe 10 connected to a service loop 15which, like analytical line 30, preferably comprises a flexible conduitmade of transparent Teflon or like material. Liquid supply 20 is locatedadjacent probe 10 and generally comprises a plurality of containers forholding and supplying the various liquids to be aspirated into the probe10 including sample liquids, reagents, marker dyes, magnetic particlesuspensions, buffers and washes.

Thus, the test packages just described are created in one or more cyclesinvolving the alternate aspiration of segments of liquid from the liquidsupply 20 and segments of air. In particular, with respect to thecreation of a sandwich type immunoassay test package, during each cycle,two liquid segments, L₁ and L₂, and three air segments, A₁, A₂ and A₃,are alternatively aspirated into the probe 10 and up into the serviceloop 15 by operation of aspiration pump 25, which is selectively coupledto the service loop 15 through transfer line 35 and shear valve 40, tobe described below, to create a test package component shown in FIG. 4.The order of aspiration during each cycle to create a test packagecomponent is as follows: A₃, L₂, A₂, L₁, A₁. Thus, a sandwich typeimmunoassay test package as shown in FIG. 2 is created by aspiratingthree successive test package components, designated as TPC-1, TPC-2 andTPC-3 in FIG. 2, in three successive cycles. In cycle 1, TPC-1 isaspirated wherein L₂ is the mixture of sample and first and secondreagents S/R₁/R₂ and L₁ is the magnetic particle suspension MP. In cycle2, TPC-2 is aspirated wherein L₂ is the second wash W₂ and L₁ is thefirst wash W₁. In cycle 3, TPC-3 is aspirated wherein L₂ is the markerdye D and L₃ is the combination of third and fourth reagents R₃/R₄. FIG.5 illustrates this creation in three cycles. As shown in FIG. 5,adjacent test package components always abut one another at an A₁/A₃interface. The chemistry test package, on the other hand, is simplycreated in one cycle by aspirating a third air segment A₃, then thebuffer B, then a second air segment A₂, then the mixture of sample andfirst reagent S/R₁, then the vanish bubble VB, then the second reagentR₂, and finally a first segment A₁.

In addition, as described in the 147 and '497 patents, an isolationliquid is aspirated into the probe 10 each time a liquid is aspirated.The isolation liquid preferably comprises an appropriate fluorocarbon orsimilar liquid, referred to herein at times as oil, which is immisciblewith the liquid supplied by liquid supply 20 and wets the hydrophobicinner walls of the system components. As a result, the system componentinner walls are coated with an isolation liquid layer whichsubstantially prevent contact therewith by the liquid and the adhesionof the same thereto.

Once the test packages are created, they are transferred to theanalytical line 30 for subsequent analysis in the following manner. Foran immunoassay using the system shown in FIG. 1B, the probe 10 andservice loop 15 is capable of holding a maximum of two test packagecomponents. As each successive test package component making up a testpackage is aspirated into probe 10, the previously aspirated testpackage component is moved towards the shear valve 40. The service loop15 is selectively connected to transfer line 35, also preferablycomprising a flexible conduit made of transparent Teflon or likematerial, through shear valve 40. Thus, when the service loop 15 isfull, i.e., contains two test package components, and the nextsuccessive test package component is aspirated by probe 10, the testpackage component contained within the service loop 15 nearest the shearvalve 40 will move into the transfer line 35. The shear valve 40 is thenactuated so as to align the transfer line 35 with the analytical line 30and, by operation of the aspiration pump 25, the test package componentcontained within the transfer line 35 is inserted into the analyticalline 30. The shear valve 40 is then actuated again so as to realigntransfer line 35 with service loop 15 and the next test packagecomponent is aspirated. Thus, for each immunoassay test package shown inFIG. 2, it takes three complete actuations of shear valve 40, i.e., acomplete actuation meaning from aligning the transfer loop 35 with theservice loop 15 to aligning the transfer loop 35 with the analyticalline 30 and back again, to move the complete immunoassay test package,one test package component at a time, into the analytical line 30. Itwill be appreciated by one of skill in the art that if an immunoassaytest package containing eight liquid segments as described above isused, it will take four complete actuations of shear valve 40 to movethe complete immunoassay test package into the analytical line 30.

For a chemistry analysis using the system shown in FIG. 1A, the transferis virtually identical except that the probe and service loop arecapable of holding two complete chemistry test packages. Accordingly,once the probe and service loop are full, as each successive testpackage is aspirated (in a single cycle as described above), the testpackage nearest the shear valve 40 moves into the transfer line 35.Thus, unlike the immunoassay system shown in FIG. 1B which requiresthree actuations of the shear valve 40 to move a single complete testpackage to the analytical line 30, in the chemistry system shown in FIG.1A, each actuation of the shear valve 40 causes a complete chemistrytest package to be inserted into the analytical line 30. Accordingly, byoperation of the probe 10, service loop 15, aspiration pump 25, transferline 35 and shear valve 40, appropriately configured test packages arecreated and ultimately inserted into analytical line 30 for analysis.

Furthermore, for both systems shown in FIGS. 1A and 1B, each time theshear valve 40 is actuated to realign the transfer line 35 with theservice loop 15 as described above, stream line 70 is aligned withanalytical line 30 and stream pump 46 causes the stream of test packagesalready contained in the analytical line 30 to flow a predeterminedamount or distance in the forward and reverse directions as described indetail in the '147 patents and '497 patents. This flow causes the testpackages to successively move through the system and, in turn, undergoappropriate analysis.

Although the preferred embodiment of the present invention utilizesshear valve 40, any inlet valve that is capable of selectivelyconnecting service loop 15, transfer line 35 and analytical line 30could be used, such as a three-way valve.

As will be known by one of skill in the art, in a typical so-calledsandwich immunoassay analysis, the sample S is allowed to react withfirst and second reagents R₁ and R₂ within the test package for aparticular, fixed period of time as defined by the assay protocol. Then,the magnetic particles are transferred out of the magnetic particlesuspension MP and into the S/R₁/R₂ segment, where they are allowed tomix for an additional specified amount of time as defined by the assayprotocol. Thereafter, the magnetic particles are separated from theS/R₁/R₂ segment, are transferred to and washed in washes W₁ and W₁, andare transferred to and reacted with a combination of third and fourthreagents R₃/R₄. The reaction between the magnetic particles and thecombination of third and fourth reagents R₃/R₄ generates a detectableresponse in the form of photometric signals which are in proportion tothe analyte concentration in the sample S. As noted above, a suitableapparatus for carrying out such an immunoassay is shown in FIG. 1B andincludes first and second magnets 60 and luminometer 65. The firstmagnet 60 is used to transfer the magnetic particles into the S/R₁/R₂segment after a specific amount of time, and the second magnet 60 isused to transfer the magnetic particles from the S/R₁/R₂ segment intowashes W₁ and W₂ and ultimately into the R₃₁R₄ segment. The luminometer65 is used to detect and measure the photons emitted from the segmentR₃/R₄ containing the magnetic particles.

Another well known type of immunoassay is the so-called competitiveimmunoassay. In the test package for a competitive immunoassay, amixture of sample and a first reagent S/R₁ are separated from a secondreagent R₂ by a vanish bubble VB. Thus, the sample S and first reagentR₁ are preincubated in the analytical line 30 for a fixed period of timebefore being mixed with the second reagent R₂ through operation of thevanish zone 50. Then, the remainder of the method proceeds as describedabove in connection with the sandwich method.

As will be known by one of skill in the art, in a typical chemistryanalysis a mixture of the sample and a first reagent SIR, are separatedfrom a second reagent R₂ by a vanish bubble VB. Thus, the sample S andfirst reagent R₁ are pre-incubated in the analytical line 30 for a fixedperiod of time before being mixed with the second reagent R₂ throughoperation of the vanish zone 50. The chemical reaction between thesample S, reagent R₁ and reagent R₂ produces a chromofore which absorbslight at a specific wavelength. The light absorption is measured everytime a S/R₁/R₂ segment passes through the Flow Cells 45. Since the lightabsorption is proportional with the concentration of chromofore, whichin turn depends on the amount of the analyte in the sample, the analyteconcentration can be determined.

In the systems just described, it is very important to keep the timingof the various analyses, whether it be the chemistry or the immunoassay,very accurate and uniform. In particular, in the chemistry method it isimportant that all test packages pass the vanish zone and the flow cellsat precise and uniform times, and in the immunoassay method it isimportant that all test packages reach the first magnet and the secondmagnet (where the magnetic particles are removed from the S/R₁/R₂segment, thereby terminating the immunochemical reactions) at veryprecise and uniform times. In order to achieve and maintain such precisetiming, it is necessary for the test packages to be maintained at auniform and consistent length.

The uniformity among test packages can be adversely effected during theaspiration thereof in the following manner. When a liquid with a highsurface tension, such as serum, is aspirated, the thickness of theisolation liquid film in the probe 10 and service loop 15 decreases anda portion of the isolation liquid is displaced and pushed ahead of thesegment with the high surface tension. The displacement of oil resultsin a change in the volume of the probe 10 and service loop 15. As aresult, shearing would not occur in the middle of the A₁/A₃ segment,which normally causes the insertion of small air segments into theanalytical line 30, which can result in a substantial decrease in thelength of the stream.

In addition, in connection with an immunoassay, it is extremelyimportant for the glowing segment, i.e., the R₃/R₄ segment mixed withthe magnetic particles that is emitting photons, to have a constantvelocity as it passes through the luminometer 65. If the velocity is notconstant, the dwell time, meaning the length of time the glowing segmentspends in the luminometer read head, will vary, and the number ofphotons counted will be incorrect. For example, if the speed of thestream is lower than it should be, the dwell time will be higher, andmore photons will be counted. As a result, the measured light intensitywill be overestimated and the results will be distorted.

Moreover, in connection with a chemistry analysis, it is generallydesirable to take measurements on a specific predetermined number oftest packages at the first flow cell 45 during the forward flow of thestream of test packages in the analytical line 30. If the length of thetest packages, and thus the stream, becomes too large, less testpackages will be observed during the forward flow of the stream for agiven movement of the stream pump 46. If the length of the test packagesbecomes too small, more test packages will be observed during theforward flow of the stream and there is a higher probability forundesirable merging of adjacent liquid segments inside the vanish zone50. In addition, if the length of the stream is permitted to vary, thetiming at which the test packages reach the flow cells 45 will be thrownoff. It is important that the timing be accurate because the accuracy ofthe measurements to be taken depends on the state of the reactionstaking place in the test packages, which in turn depends on the reactionrate.

SUMMARY OF THE INVENTION

The present invention relates to a method and apparatus for controllinga stream of liquid and air segments wherein the liquid and air segmentsare selectively aspirated into a first fluid conduit in a plurality ofcycles, each cycle beginning with the aspiration of a first air segmentand ending with the aspiration of a final air segment. The first andfinal air segments each have a volume. The liquid and air segments arethen transferred from the first fluid conduit to a second fluid conduit.The volume of the final air segment of each cycle is adjusted after thefinal air segment has moved into the second fluid conduit. Next, theliquid segments and the air segments of each cycle are transferred fromthe second fluid conduit to a third fluid conduit. The volume of thefirst air segment of each cycle is then adjusted after the first airsegment has moved into the third fluid conduit.

One aspect of the present invention is directed to an apparatus forcontrolling a stream of liquid packages, each liquid package comprisinga plurality of liquid and air segments. The apparatus includes a probefor selectively aspirating liquid segments and air segments into a firstfluid conduit in a plurality of cycles wherein each cycle ends with theaspiration of a final liquid segment and a final air segment in thatorder. The apparatus further includes a second fluid conduit and a valvecoupled to the first fluid conduit and the second fluid conduit. Thevalve is adapted to be actuated between a first position in which thesecond fluid conduit is coupled to the first fluid conduit and a secondposition in which the second fluid conduit is not coupled to the firstfluid conduit. An air and liquid interface detector is positioned alongthe second fluid conduit a fixed distance from the valve, the fixeddistance corresponding to a predetermined fixed volume within the secondfluid conduit. The apparatus includes means for stopping the aspirationof the final air segment in a current cycle when the air and liquidinterface detector detects an interface between an air segment and aliquid segment of a previous cycle which have moved into the secondfluid conduit after the final liquid segment of the previous cycle hasfully entered the second fluid conduit and for actuating the valve fromthe first position to the second position at a time subsequent to thisdetection.

A further aspect of the present invention is directed to a method forcontrolling a stream of liquid packages wherein each liquid packagecomprises a plurality of liquid and air segments. The method includesselectively aspirating liquid segments and air segments into a firstfluid conduit in a plurality of cycles, each cycle ending with theaspiration of a final liquid segment and a final air segment in thatorder. The first fluid conduit is coupled to a valve and the valve iscoupled to a second fluid conduit. The valve is adapted to be actuatedbetween a first position in which the first fluid conduit is coupled tothe second fluid conduit and a second position in which the first fluidconduit is not coupled to the second fluid conduit. The method furtherincludes detecting an interface between an air segment and a liquidsegment of a previous cycle which have moved into the second fluidconduit at a position along the second fluid conduit which is a fixeddistance from the valve after the final liquid segment of the previouscycle has fully entered the second fluid conduit. The fixed distancecorresponds to a predetermined fixed volume within the second fluidconduit. The method also includes stopping the aspiration of the finalair segment in a current cycle when the interface has been detected andactuating the valve from the first position to the second position at atime subsequent to this detection.

A further aspect of the present invention is directed to an apparatusfor controlling a stream of liquid packages wherein each liquid packagecomprises a plurality of liquid and air segments. The liquid packagesare formed in one or more cycles of aspiration of liquid and airsegments, each cycle beginning with the aspiration of a first airsegment and a first liquid segment. The apparatus includes a fluidconduit into which the liquid and air segments aspirated in each of thecycles are inserted and in which the stream of liquid packages isrepeatedly bi-directionally flowed in a forward and reverse direction.The apparatus further includes a valve coupled to a first end of thefluid conduit and an air and liquid interface detector positioned alongthe fluid conduit adjacent the valve. Also provided are means forstopping the flow of the stream of liquid packages in the reversedirection at a point in time when the air and liquid interface detectordetects an interface between the first air segment and the first liquidsegment most recently inserted into the fluid conduit adjusted by adelay, wherein the delay is normalized around a predetermined nominalcenter point delay according to a feedback loop.

A still further aspect of the present invention relates to a method forcontrolling a stream of liquid packages, each liquid package comprisinga plurality of liquid and air segments. The method includes selectivelyaspirating liquid segments and air segments in a plurality of cycles,each cycle beginning with a first air segment and a first liquidsegment, and inserting the liquid segments and the air segmentsaspirated in each of the cycles into a fluid conduit having a first endsuch that for each of the cycles the first liquid segment and the firstair segment are the next-to-last and the last liquid and air segment,respectively, inserted. The method further includes flowing the streamof liquid packages in a forward direction and a reverse direction in thefluid conduit and detecting an interface between the first air segmentand the first liquid segment most recently inserted into the fluidconduit at a reference location along the fluid conduit adjacent thefirst end of the fluid conduit when the stream is flowing in the reversedirection. The flow of the stream is stopped at a point in time when theinterface is detected adjusted by a delay, the delay being normalizedaround a predetermined nominal center point delay according to afeedback loop.

According to still a further aspect of the present invention, thefeedback loop described above is based upon a time difference TD equalto T₂−T₁, wherein T₁ is a time at which a first particular liquidsegment of a particular liquid package reaches a reference point duringthe flow of the stream in the forward direction and T₂ is a time atwhich a second particular liquid segment of the particular liquidpackage reaches a reference point during the flow of the stream in thereverse direction. In an aspect of the invention, the delay iscalculated according to the following formula:Delay=CP−Error*K,wherein CP is the predetermined nominal center point delay, Error isequal to a predetermined set point minus TD, and K is a gain factor.

According to still a further aspect of the present invention, thefeedback loop is based upon a difference between a target liquid packagelength and an average length, wherein the liquid and air segmentsaspirated in each of the cycles and inserted into the fluid conduit eachcomprise a liquid package having a length, wherein the length ismeasured for a plurality of the packages during the flow of the streamin either the forward or reverse direction and wherein an average lengthis calculated using a plurality of the measured lengths. In an aspect ofthe present invention, the delay is calculated according to thefollowing formula:Delay=CP−Error*K,wherein CP is the predetermined nominal center point delay, Error isequal to the target liquid package length-minus the average length, andK is a gain factor.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the invention will be apparent uponconsideration of the following detailed description of the presentinvention, taken in conjunction with the following drawings, in whichlike reference characters refer to like parts, and in which:

FIG. 1A is a diagram of a prior art capsule chemistry sample liquidanalysis system configured for conducting a chemistry analysis;

FIG. 1B is a diagram of a prior art capsule chemistry sample liquidanalysis system configured for conducting an immunoassay analysis;

FIG. 2 is a diagram of a liquid test package suitable for conducting amagnetic particle-based heterogeneous immunoassay analysis;

FIG. 3 is a diagram of a liquid test package suitable for conducting achemistry analysis;

FIG. 4 is a diagram of a liquid test package component comprising aportion of the liquid test package shown FIG. 2;

FIG. 5 is a diagram showing the service loop and transfer line of thesystem shown in FIG. 1B which illustrates the creation of the liquidtest package shown in FIG. 2 in three cycles;

FIG. 6A is a diagram of a capsule chemistry sample liquid analysissystem according to an aspect of the present invention configured forconducting a chemistry analysis.

FIG. 6B is a diagram of a capsule chemistry sample liquid analysissystem according to an aspect of the present invention configured forconducting an immunoassay analysis;

FIG. 7 is a diagram showing the transfer line, shear valve and transferline bubble detector of the system shown in FIG. 6B according to anaspect of the present invention;

FIG. 8 is a diagram showing the probe, service loop, transfer line,shear valve and transfer line bubble detector of the system shown inFIG. 6B according to an aspect of the present invention whichillustrates the creation of the liquid test package shown in FIG. 2;

FIG. 9 is a diagram showing the analytical line, shear valve and shearvalve bubble detector of the system in FIG. 6B according to an aspect ofthe present invention;

FIG. 10 is a diagram showing the analytical line of the system shown inFIG. 6B which illustrates a read frame according to an aspect of thepresent invention;

FIGS. 11 and 12 are diagrams of the analytical line and luminometer ofthe system shown in FIG. 6B which illustrates the times at which theluminometer starts and stops counting photons.

DETAILED DESCRIPTION

Referring to FIGS. 6A and 6B, embodiments of the system according to anaspect of the present invention configured for conducting a chemistryanalysis and an immunoassay analysis, respectively, are shown. As shownin FIGS. 6A and 6B, transfer line bubble detector 75 is affixed alongtransfer line 35. Bubble detectors are well known in the art and arecapable of sensing an interface between an air segment and a liquidsegment in a flowing stream of air and liquid segments. Examples ofsuitable bubble detectors are described in U.S. Pat. No. 5,466,946 andU.S. application Ser. No. 08/995,738, both owned by the assignee hereof,the disclosures of which are incorporated herein by reference. As analternative to a bubble detector, other devices capable of detecting aninterface between an air segment and a liquid segment could be usedwithout departing from the scope of the present invention. For example,a conductivity sensor which detects the conductivity within a fluidconduit when a liquid is present therein could be used.

FIG. 7 is a diagram showing transfer line 35, shear valve 40 andtransfer line bubble detector 75 of FIGS. 6A and 6B. For illustrativepurposes, the function of the transfer line bubble detector 75 will bedescribed in connection with a sandwich type immunoassay analysis. Thus,in FIG. 7, transfer line 35 has inserted therein a test packagecomponent as described above in connection with FIG. 4 comprising A₃,L₂, A₂, L₁ and A₁. It should be understood, however, that theconfiguration and operation described herein is not to be limited tosystems performing only immunoassay analyses, but rather would applyequally to systems performing other analyses such as the chemistryanalysis described above. As will become apparent below, the onlylimitation to the application of the transfer line bubble detectorconcept to be described herein is that it would not be applicable totest package components in which the sample is in the first test packagecomponent adjacent to the shear valve 40 in FIG. 7, such as a serologymethod.

As shown in FIG. 7, according to an embodiment of the present invention,the transfer line bubble detector 75 is positioned a fixed distance fromthe shear valve 40 equivalent to the combined volume of L₁ and A₁ of thetest package component. In appropriate circumstances, the positioning ofthe transfer line bubble detector 75 may be adjusted for a system delayin stopping the aspiration pump 25. The fixed distance corresponds to afixed volume within the transfer line 35 equal to the sum of thepreferred volumes of the L₁ and A₁ segments. According to a preferredembodiment of the present invention, in the test package componentswhich make up the sandwich type immunoassay test package, the volume ofA₂ is between 15 and 20 μl due to the tolerance on the volume of theaspiration syringe of probe 10, and most preferably 14 μl, the volumesof A₁ and A₃ are 7 μl each, and the volumes of L₁ and L₂ are 28 μl each.Thus, in this preferred embodiment, the sum of A₁ and A₃ is always 14μl.

As noted above, it is important to keep the volumes of the A₁ and A₃ ofeach test package component constant, preferably 7 μl, because it isimportant to keep the volume, and consequently the length, of each testpackage component and thus the length of each test package uniform inorder to maintain accurate timing as the stream of test packages movesthrough the system. In the preferred embodiment of the present inventionfor conducting an immunoassay analysis, the first magnet 60 inanalytical line 30 must transfer the magnetic particles from themagnetic particle suspension MP into S/R₁/R₂ segment 13 minutes afterinsertion into the analytical line 30 and the second magnet 60 inanalytical line 30 must transfer the magnetic particles out of theS/R₁/R₂ segment at 19 minutes after inserting into the analytical line30. However, as discussed above, the length of the test packagecomponents can be adversely affected when a liquid with a high surfacetension is aspirated into probe 10. This adverse effect may throw offthis critical timing.

Thus, referring to FIGS. 7 and 8, this problem is solved according to anaspect of the present invention by controlling or altering the volume ofthe A₁ segment of the test package component that is located in thetransfer line 35 and that is about to be inserted into the analyticalline 30 (TPC-1 in FIG. 8). Specifically, as noted above, adjacent testpackage components always abut one another at an A₁/A₃ interface. Thevolume of the A₁ segment in the transfer line 35 is controlled oraltered by controlling the point at which the A₁/A₃ interface is shearedby the shear valve 40 as the test package component is moved into thetransfer line 35. According to an aspect of the present invention, theA₁/A₃ interface is sheared based on the transfer line bubble detector 75detecting the interface between the A₂ and L₁ segments of the testpackage component as that test package component is moved into thetransfer line 35 from the service loop 15. When this interface isdetected, the aspiration pump 25 stops aspirating the current segment ofthe current test package component being created (TPC-3 in FIG. 8),which, as will be apparent, and as is shown in FIG. 8, will be the A₁segment of that test package component. Then, at some subsequent time,the shear valve 40 is actuated to align the transfer line 35 with theanalytical line 30. This operation, i.e., stopping aspiration, may causethe A₁ segment of the current segment pair, meaning adjacent liquid andair segments, being created to be smaller or larger than the preferredvalue. Thus, in order to compensate, the A₃ segment aspirated in thenext cycle will be aspirated to a volume equal to 14 μl minus the volumeof A₁, thereby resulting in a total A₁ and A₃ volume equal to thepreferred volume of 14 μl. The above-described operation oftransfer-line bubble detector 75 results in test packages in which theA₁ and A₃ air segments are of a uniform specific volume. Also, A₂, L₂and L₁ are of uniform specific volumes and are accurately metered by theaspiration pump 25.

Alternatively, the transfer line bubble detector 75 could be positionedalong the transfer line 35 such that the fixed distance corresponds to afixed volume equal to the preferred volume of the A₁ segment. Thus, inthis configuration, the A₁/A₃ interface will be sheared based on thedetection of the interface between the L₁ and A₁ segments in thetransfer line 35. Other variations of the placement of the transfer linebubble detector and thus variations on the above-described fixed volumeare possible and will be apparent to one of skill in the art. Regardlessof the placement of the transfer line bubble detector 75, it alwaysdetects an appropriate liquid and air interface after the L₁ segment hasbeen fully inserted into the transfer line 35.

All of the necessary control described above is provided by appropriatesoftware, the details and specific implementations of which would bereadily apparent to one of skill in the art and thus will not bedescribed herein. The software is stored on a hard disk and is loadedinto a data acquisition computer at startup.

Furthermore, as noted above, in the case of an immunoassay analysis, itis important for the glowing segment, i.e. the R₃/R₄ liquid segmentcontaining magnetic particles that is emitting photons, to have aconstant velocity as it passes through the luminometer 65 placed alonganalytical line 30. A constant velocity can be achieved by maintaining afixed distance between the position of the glowing segment and theluminometer 65 at the start of the forward stream motion during whichglowing segment moves past the luminometer 65. The range of variation ofthe position of the glowing segment at the start of the forward streamshould be no more than ±1 segment. To achieve a positional stability of±1 segment, the entire length of the air and liquid segments in theanalytical line 30 from the shear valve 40 to the second magnet 60 mustnot vary by more than ±0.5%.

In order to provide this stability and thus maintain the constantvelocity described above, as shown in FIG. 6B, the system according toan aspect of the present invention includes shear valve bubble detector80 placed along analytical line 30 adjacent to shear valve 40 whichoperates in conjunction with a feedback loop to be described below.Preferably, the shear valve bubble detector 80 is positioned at adistance corresponding to at least 7 μl to the shear surface of theshear valve 40. The excess volume above 7 microliters is used to allowfor the distance needed by the stream to stop. As noted above, otherdevices capable of detecting an interface between a liquid segment andan air segment could be used as an alternative to shear valve bubbledetector 80.

As described above, the test package components forming immunoassay testpackages are inserted into analytical line 30 by operation of transferline 35, shear valve 40 and aspiration pump 25 such that the lastsegment to enter analytical 30 is always the A₃ segment. Also asdescribed above and in detail in the '147 and '497 patents, after eachsuccessive test package component is inserted into analytical line 30,the shear valve 40 is actuated to align stream line 70 with analyticalline 30, and by operation of stream pump 46, the stream in theanalytical line 30 is flowed a predetermined amount in first the forwardand then the reverse direction.

In order to monitor and control the length of the stream in analyticalline 30 and thus maintain the necessary fixed distance between eachglowing segment of each test package and the luminometer 65, the size ofthe A₃ segment of each new test package component inserted into theanalytical line 30 is controlled, i.e., increased or decreased. Thiscontrol and manipulation is accomplished by the shear valve bubbledetector 80, the shear valve 40, and stream vent valves 85 which, whenactivated, stop the flow of the stream in analytical line 30. Inparticular, referring to FIG. 9, as the stream is flowed in thedirection toward the shear valve 40, i.e., the reverse direction, theA₃/L₂ interface is detected by the shear valve bubble detector 80 and,after a programmed delay of up to 300 milliseconds after this detection,the stream line vent valves 85 are activated to stop the stream. Theparticular programmed delay is determined by a feedback control loop tobe described below. After the stream line vent valves 85 are activated,the shear valve 40 is actuated, thereby altering the size of the A₃segment. The resized A₃ segment is combined with the A₁ segment of thenext inserted test package component from the transfer line 35 to yieldan air segment A₁/A₃ of a controlled volume. Thus, the size of the airsegments formed at the shear valve 40 depend on the delay between thetime that the shear valve bubble detector 80 senses the A₃/L₂ interfaceof the last test package component in the returning stream, and the timethat the stream vent valves 85 are activated to stop the stream.

The feedback control loop which determines the appropriate delaydescribed above will now be described with reference to FIGS. 10 through12. The feedback control loop uses as its basis for measurement what isknown as a read frame comprising particular portions of adjacent testpackages. In particular, as shown in FIG. 10, each read frame starts atthe air-liquid interface of the R₃/R₄ segment of one test package, andends at the air-liquid interface of the W₂ segment from the nextsuccessive test package.

As shown in FIG. 11, when the beginning of the read frame reaches thebubble detector 55 closest to luminometer 65 in FIG. 6B during theforward flow of the stream, the luminometer 65 begins counting photonsand the time is recorded. As shown in FIG. 12, when the end of the readframe reaches the bubble detector 55 during the forward flow of thestream, the luminometer 65 stops counting photons and the data is saved.After the five liquid segments comprising the read frame have passedthrough the luminometer in the forward direction (left to right in FIGS.11 and 12), the stream flow stops and reverses direction. When the endof the read frame reaches bubble detector 55 during the reverse flow ofthe stream, the luminometer 65 starts counting photons again and thetime, T₂, is recorded. When the front of the read frame reaches bubbledetector 55 during the reverse flow of the stream, the luminometer 65stops counting photons and the data is saved.

As noted above, the goal is to have the read frame, in particular theglowing segment, start far enough back from the luminometer 65 in boththe forward and reverse directions such that the stream has time toreach a constant velocity during the time in which photons are counted.This goal is accomplished according to an aspect of the presentinvention by controlling the stream start position in such a way thatthe time differential TD, equal to T₂ minus T₁, remains close to apredetermined set point. In a preferred embodiment of the presentinvention, the set point is equal to 3700 milliseconds. If the streamstarts forward at too great a distance from the luminometer 65 andbubble detector 55, TD will be too small. To remedy this problem, moreair will need to be added to the front end of the stream (at the shearvalve 40) to push the stream front closer to the luminometer 65. This isdone by decreasing the shear valve bubble detector delay describedabove. Similarly, if the stream starts forward at too little a distancefrom the luminometer 65, TD will be too large. To remedy this problem,the stream front will need to be moved away from the luminometer 65 bydecreasing the air at the front end of the stream using an increasedshear valve bubble detector delay.

The appropriate control is accomplished by means of a proportionalintegral differential control loop where only the proportional elementof control is used. In such a control loop according to an aspect of thepresent invention, the shear valve bubble detector delay is calculatedaccording to the following equation:Delay=CP−Error*K,wherein CP is the nominal value of the delay which results in airsegments of nominal or normal size, which will vary depending on theparticulars of the analytical line 30, Error equals the set point,described above, minus TD, and K is a gain factor. In a preferredembodiment of the present invention, the nominal value of the delay CPis equal to 200 milliseconds, which results in air segments of about 17μl, and K is equal to 0.5. Furthermore, the value of Error is limited tofrom −200 to +200 milliseconds. Error values outside of this range arenot expected and are eliminated in order to prevent excessive delayadjustments. Thus, as will be apparent, the shear value bubble detectordelay is normalized to +100 milliseconds around the control range centerpoint CP.

In addition, as noted above, in the case of a chemistry analysis, thegoal is to take measurements on a specific predetermined number of testpackages, preferably 13, at the first flow cell 45 during each forwardflow of the stream of test packages in the analytical line 30. In orderto achieve this goal, which depends on controlling the length of thetest packages and thus the length of the stream, a feedback loop,similar to the one described above in connection with an immunoassayanalysis, which operates in conjunction with shear valve bubble detector80 is used. In particular, this feedback control loop controls the delaybetween the time at which the shear valve bubble detector 80 detects theinterface between the A₃ and B segments of the last test package in thereturning stream and the time at which the stream vent valves 85 areactivated to stop the stream. Thus, the size of each new A₃ segmentinserted into the analytical line 30 can be controlled.

In the chemistry feedback loop, instead of measuring TD equal to T₂minus T₁, the length of a set of test packages, measured from buffer Bto buffer B, is measured during the forward or reverse motion of thestream in the analytical line 30. The length of each test package isactually measured in terms of the transit time of the test package pastone of the flow cells 45, preferably the first flow cell 45.Facilitating this function is the fact that each flow cell 45 isprovided with the capability to detect liquid to air interfaces muchlike a bubble detector. Thus, the set of test packages whose lengths aremeasured comprises all of those test packages that pass the particularflow cell 45 during either the forward or reverse motion of the stream,whichever the case may be. An average test package length, Taver, isthen calculated using the measured lengths. In a preferred embodiment,only the measured lengths of certain of the test packages that satisfycertain criteria to be described below are used to calculate Taver. Thisaverage is then compared to a target test package length, Tset. With anaverage test package length equal to Tset, the correct number of testpackages will be measured by the first flow cell 45. If the average testpackage length is too large, it is necessary to reduce the size of theA₃ segment of the last test package inserted into the analytical line 30by increasing the delay described above. If the average test packagelength is too small, it is necessary to increase the size of the A₃segment of the last test package inserted into the analytical line 30 bydecreasing the delay described above.

As noted above, in the preferred embodiment of the present invention,not every measured length is used to calculate Taver. Instead, only themeasured lengths of those test packages that satisfy certain criteriaare used. The particular criteria used can vary. One example is to useonly test packages whose S₁/R₁ and R₂ segments have merged in the vanishzone 50, so-called “merged” packages. Another example is to use onlytest packages whose S₁/R₁ and R₂ segments have not yet merged in thevanish zone 50, so-called “non-merged” packages. It will be appreciatedby one of skill in the art that in order to accurately identify a testpackage as “merged” or “non-merged”, it will be necessary to use anappropriate pattern recognition protocol and that such a protocolrequires that the test package be moving at a uniform, predeterminedvelocity. If it is determined that the test package is not moving at theuniform, predetermined velocity, then the test package cannot beidentified and will thus not be used in the calculation of Taver. Aswill be apparent to one of skill in the art, one way of determiningwhether the test package is moving at the uniform, predeterminedvelocity is to measure the ratio of the transit times of two adjacentliquid segments within the test package and then compare that ratio toan expected value for test packages moving at the uniform, predeterminedvelocity.

Another criteria that can be used, and that is in fact used in apreferred embodiment of the present invention, is to check whether themeasured length of each test package falls within the range of an upperlimit and a lower limit. This test thus checks whether each measuredlength is a reasonable, expected value, and results in only reasonablemeasurements being used in the calculation of Taver. Based on empiricaldata, it has been determined that the preferred upper limit is 300milliseconds and the preferred lower limit is 160 milliseconds. If themeasured length in terms of transit time falls outside of this range, itwill not be used to calculate Taver.

In a preferred embodiment of the present invention which utilizes theparticular criteria just described, typically nine “merged” testpackages are used to calculate Taver. It should be understood, however,that other appropriate criteria can be used to determine which testpackage lengths are used to calculate Taver without departing from thescope of the present invention. For example, both “merged” and“non-merged” test packages could be used.

According to an aspect of the present invention, the delay for thechemistry feedback loop is calculated according to the followingequation:Delay=CP−Error*K,wherein CP is as described above with respect to the immunoassayfeedback loop, Error equals Tset−Taver, and K is a gain factor. In apreferred embodiment of the present invention, Tset equals 220milliseconds and K equals 0.5.

While in the preferred embodiments described herein only theproportional element of control is used, it will be apparent to one ofskill in the art that the integral or differential elements of controlcould also be used.

All of the necessary control for the system components described aboveis provided by appropriate software loaded into the data acquisitioncomputer. The details and particular implementations of the softwarewould be readily apparent to one of skill in the art based on thefunctions described above and thus will not be repeated herein.

One skilled in the art will appreciate that the present invention can bepracticed by other than the described embodiments, which are presentedfor purposes of illustration and not of limitation.

1. A method for maintaining a uniform and consistent length of aplurality of liquid test packages each comprising a plurality of testpackage components in a capsule chemistry sample liquid analysis systemby ensuring that each test package, which comprises alternating segmentsof liquid and air, has a predetermined volume, comprising: (a)selectively aspirating into a first fluid conduit the plurality of testpackages in successive cycles, wherein each test package is aspirated ina separate cycle that begins with a first air segment having a first airvolume, and ends with a final liquid segment and a final air tin thatorder, and wherein each final air segment has a final air volume; (b)transferring in sequence the test package from each cycle from saidfirst fluid conduit to a second fluid conduit; (c) controlling oraltering the volume of the final air segment of each test package as itenters the second fluid conduit; (d) transferring each test package fromsaid second fluid conduit to a third fluid conduit; (e) controlling oraltering the volume of the first air segment of each test package as itenters the third fluid conduit.
 2. The method of claim 1, wherein instep (c) the volume of the final air segment in the second fluid conduitis controlled or altered by detecting an interface between the finalliquid segment and an adjacent air segment that is not the final airsegment at a reference location along the second fluid conduitcorresponding to a predetermined volume equal to the sum of thepreferred volume of the final air segment and the final liquid segment.3. The method of claim 2, wherein the reference location is a fixeddistance along the second fluid conduit that corresponds to apredetermined fixed volume within the second fluid conduit.
 4. Themethod of claim 3, wherein the volume of the final air segment in thesecond fluid conduit is controlled or altered by shearing the final airsegment of the test package at the interface of the first air segment ofthe adjacent test package by stopping the aspiration of a test packagecomponent currently being aspirated into the first fluid conduit.
 5. Themethod of claim 1, wherein in step (e) the first air segment enters thethird fluid conduit adjacent to a first liquid segment, such that thefirst liquid segment and the first air segment are the next-to-last andlast liquid and air segments, respectively, entering the third fluidconduit.
 6. The method of claim 5, wherein the volume of the first airsegment in the third fluid conduit is controlled or altered with a valveafter detecting an interface between the first air segment and the firstliquid segment at a reference location along the third fluid conduit,and stopping the flow of the test package in the third fluid conduit. 7.The method of claim 6, wherein each test package in the third fluidconduit flows a predetermined distance, first in the forward directionand then in the reverse direction, in order to detect the interfacebetween the first air segment and the first liquid segment.
 8. Themethod of claim 7, wherein the interface is detected when the testpackage flows in the reverse direction.
 9. The method of claim 6,wherein the valve controls or alters the volume of the first air segmentafter a predetermined time delay.